Ginger Withers (2011) CIL:12550, Rattus, multipolar neuron. CIL. Dataset

Presynaptic contacts are abundant on both cell bodies and the dendritic arbor of cultured hippocampal neurons after 15 days in vitro. Neurons were immunostained for MAP2, a microtubule associated protein localized to dendrites (green) and synapsin I (red), a presynaptic vesicle protein. With the exception of the immunostained presynaptic terminals, axons are not visible in this preparation. A relatively low power objective (20X) was used to capture the entire dendritic arbor of individual cells.
Detailed methods: Embryonic rat hippocampal neurons were prepared as previously described (see Kaech and Banker, 2006, Nat Protoc). Cells were prepared for fluorescent staining as previously described (Withers and Banker, 1998, in Culturing Nerve Cells, MIT Press). Briefly, cells were fixed (4% formaldehyde, 4% sucrose in phosphate buffered saline, pH 7.4), permeabilized with 0.25% Triton and immunostained for MAP2 (monoclonal HM2, Sigma, with Alexa 488 conjugated secondary, excitation, 494, emission, 519 [Invitrogen, Molecular Probes]) and synapsin I (from P. DeCamilli, with DyLight549 conjugated secondary, excitation, 555, emission, 568, [Jackson Immunoresearch]). Images were acquired with a Leica DMRA microscope with a 20X (HC Fluotar, NA 0.5) lens, Photometrics CoolSnap ES CCD camera and MetaMorph software. Individual images of each fluorophore were colorized and assembled as a a stack file using MetaMorph software.

在海马神经元体外培养15天后，其细胞体和树突分支上均存在大量的突触前联系。神经元经过MAP2（一种定位于树突的微管相关蛋白，呈绿色）和突触素I（一种突触前囊泡蛋白，呈红色）的免疫染色。除免疫染色的突触前终端外，本制备中轴突不可见。采用较低倍率物镜（20倍）以捕捉单个细胞的整个树突分支。详细方法：如前所述（参见Kaech和Banker，2006，Nat Protoc），制备胚胎大鼠海马神经元。细胞制备荧光染色方法如前所述（Withers和Banker，1998，在《培养神经细胞》一书中，MIT Press）。简而言之，细胞用4%甲醛、4%蔗糖磷酸盐缓冲液（pH 7.4）固定，用0.25%的Triton处理以实现通透化，并分别进行MAP2（单克隆HM2，Sigma，与Alexa 488偶联的二级抗体，激发波长494，发射波长519 [Invitrogen，Molecular Probes]）和突触素I（来自P. DeCamilli，与DyLight549偶联的二级抗体，激发波长555，发射波长568，[Jackson Immunoresearch]）的免疫染色。图像使用Leica DMRA显微镜配备20倍（HC Fluotar，NA 0.5）镜头、Photometrics CoolSnap ES CCD相机和MetaMorph软件获取。每个荧光体的单个图像经过着色并使用MetaMorph软件组装成一个堆叠文件。